Clinical and Genetic Heterogeneity in Progressive External Ophthalmoplegia Due to Mutations in Polymerase γ FREE

PROGRESSIVE EXTERNAL ophthalmoplegia (PEO) with multiple mitochondrial DNA (mtDNA) deletions in muscle can be inherited as a dominant or a recessive trait.^1- 2 Clinical manifestations are heterogeneous and are usually more variable and severe in the recessive forms.² Besides PEO, clinical features include deafness, cataracts, depression, dysphagia, hypogonadism, neuropathy, and sensory ataxia.³ Muscle biopsy results typically show ragged red fibers and cytochrome c oxidase–negative fibers.³

Autosomal dominant PEO (adPEO) has been linked to mutations in genes encoding adenine nucleotide translocator-1 (ANT1) and Twinkle, a putative mtDNA helicase, or chromosome 10 open reading frame 2 (C10orf2). Both protein products are involved in mtDNA maintenance.^4- 5 Mutations in a third gene, polymerase γ (POLG), encoding the α subunit of mtDNA polymerase γ, were identified in patients with either adPEO or autosomal recessive PEO (arPEO), which is often associated with multisystemic disorders.^6- 8 In this study, we screened the coding region of POLG in 30 patients with PEO and multiple mtDNA deletions to identify mutations, investigate their frequency, and assess phenotype-genotype correlations.

Thirty patients (16 men and 14 women) with PEO and multiple mtDNA deletions in muscle were included in this study. Twenty-one patients had complex clinical diagnoses, including neuropathy, ataxia, psychiatric disorders, deafness, and cataracts. Twenty-three patients (77%) had affected relatives, and the other cases were sporadic. None had mutations in ANT1 and C10orf2. In this article we describe the clinical features in only the 4 patients with POLG mutations.

Patient 1 (Figure 1A), a 51-year-old Ukrainian woman, was born to nonconsanguineous parents. She was healthy until age 4 years, when she was noted to have a learning disability. Mental retardation was later diagnosed, and she required special schooling throughout childhood. At age 41 years, she had difficulty moving her eyes, and she had to turn her head to fixate on objects. She also developed progressive bilateral ptosis. She did not have dysarthria, dysphagia, dyspnea, or limb weakness. Both her father, who died at age 72 years, and her paternal grandmother, who died at age 93 years, had long-standing histories of bilaterally impaired eye movements. The patient's brother and his 7-year-old daughter are healthy. Neurological examination results showed bilateral ptosis, despite previous blepharoplasties, and minimal lateral and convergence eye movements. No other neurological abnormalities were noted. Endocrinological study results, including parathyroid and thyroid function test results, were normal. Magnetic resonance imaging of the brain demonstrated mild dilatation of the subarachnoid spaces, prominence of the cerebellar folia, and a retention cyst along the posterior wall of the sphenoid sinus.

Patient 2 (Figure 1B), a 61-year-old man, noted numbness in his legs at age 47 years. Later, he developed PEO, generalized muscle weakness, exercise intolerance, unsteadiness (especially in the dark or with eyes closed), abdominal cramping, and gastrointestinal dysmotility with alternating diarrhea and constipation. A 63-year-old sister had an 18-year history of PEO, exercise intolerance, distal limb weakness, and diabetes mellitus. She also had peripheral neuropathy with stocking-glove numbness. Her 44-year-old son and a 42-year-old daughter have diabetes mellitus. The proband's parents are described as healthy, although the mother apparently had mood swings. A maternal aunt had PEO.

Patient 3 (Figure 1C) was a 43-year-old man, who, at age 7 years, developed progressive hearing loss. At age 10 years, he had difficulty with his balance, and at age 25 years, he had dysconjugate gaze. At age 31 years, he had hypogonadism with low testosterone levels and normal pituitary function. Neurological examination results showed impaired communication and speech because of severe deafness and oropharyngeal muscle weakness. In addition, he had bilateral ptosis and ophthalmoparesis, with restriction of eye movement in all directions to 60% of normal. There was also proximal and distal symmetrical limb weakness, more severe in the legs than in the arms, and impaired vibration sensation in the feet. Electromyograms showed mixed neuropathic and myogenic features. His 35-year-old brother has similar but more severe symptoms and is wheelchair-bound. Another brother, 2 sisters, their children, and both parents are healthy.

Patient 4 (Figure 1D), a 58-year-old woman, at age 30 years noted slowly progressive ptosis and limitation of eye movements. Later, she developed ataxia, orthostatic dizziness, cataracts, and gastrointestinal dysmotility with diarrhea and constipation. Neurological examination results showed normal mental status, ptosis with almost complete PEO, vibration sensory loss, sensory ataxia, and areflexia. Her sister, who had died in a car accident, reportedly had PEO. Her parents and her 4 children are healthy.

Routine histological studies and histochemical staining for cytochrome c oxidase and succinate dehydrogenase were performed as previously described.⁹ Respiratory chain enzyme activities in muscle in patients 2, 3, and 4 were measured as previously described.¹⁰

Total DNA was extracted from muscle by using a DNA isolation kit (Puregene; Gentra, Minneapolis, Minn), and mtDNA was analyzed with Southern blotting as previously described to check for multiple deletions.¹¹ Mutations were detected with single-stranded conformational polymorphism analysis. We used a set of primers that amplified all exons with flanking intronic sequences of POLG, except exon 1, which is not translated (Table 1). Reactions were performed in 25 µL of 10mM Tris hydrochloride (pH 8.9) containing 0.4µM each of the forward and reverse oligonucleotide primers; 1.5 mM of magnesium chloride; 0.2mM each of dATP, dGTP, and dTTP; 0.02mM of dCTP; 1 µCi (37 kBq) of α-³²P dCTP; and 1.25 units of Taq DNA polymerase (Roche, Indianapolis, Ind). Polymerase chain reaction (PCR) conditions for exons 4, 5, 6, 7, 8, 9, 10, 22, and 23 were 94°C for 5 minutes, followed by 35 cycles at 94°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, and a final extension step at 72°C for 7 minutes. For exons 2, 17, and 18, the annealing temperature was 60°C, whereas it was 64°C for exons 11, 12, 13, 14, 15, 16, 19, 20, and 21. Samples were denatured and separated on 6% mutation detection enhancement polyacrylamide gel (Cambrex BioScience Rockland Inc, Rockland, Me) with 5% glycerol, according to the manufacturer's protocol.

Conformations of single-stranded DNA were visualized with autoradiography by using BioMax film (Kodak, Rochester, NY). Samples with abnormal patterns were directly sequenced by using the ABI PRISM BigDye Terminators v3.0 Cycle Sequencing Kit and a 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif). The presence of the mutations was confirmed by means of PCR–restriction fragment length polymorphism analysis. PCR conditions were 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, annealing for 1 minute, 72°C for 1 minute, and a final extension step at 72°C for 7 minutes. Primers, annealing temperatures, and restriction endonucleases used to perform PCR–restriction fragment length polymorphism analysis are listed in Table 2.

Muscle biopsy results in all 30 patients revealed ragged red fibers and cytochrome c oxidase–negative fibers (data not shown). Biochemical analysis of muscle extracts showed decreased activities of multiple respiratory chain enzymes; values in patients with POLG mutations are listed in Table 3). All patients had multiple mtDNA deletions detected with Southern blot analysis (data not shown).

We found POLG mutations in 4 patients (13% of all cases; 17% of familial cases). In agreement with results from a previous report,⁷ we considered the A of the AUG chain-initiation codon of the POLG messenger RNA as nucleotide position +1 and the corresponding methionine as the first amino acid residue of the protein sequence.

Patient 1 was heterozygous for a G3227T transition in exon 20 of the POLG gene, which resulted in a Gly1076Val amino acid change. Analysis of DNA in her healthy brother did not show any POLG mutation (Figure 2A).

Patient 2 and his affected sister were both heterozygous for a C1760T transition in exon 10, which resulted in a Pro587Leu amino acid change (Figure 2B). Patient 3 was heterozygous for the same C1760T mutation identified in patient 2 (Figure 2C).

Patient 4 was compound heterozygous for a G2665A mutation in exon 17, which resulted in an Ala889Thr amino acid change (Figure 2D), and for a C1735T transition in exon 10, which resulted in an Arg579Trp amino acid change (Figure 2E). None of these mutations was found in 120 control alleles from patients who underwent diagnostic muscle biopsy but were ultimately deemed free of neuromuscular disease.

In these 4 patients with POLG mutations, we also found a Gln1236His change already described as a polymorphism⁷ and 2 heterozygous nucleotide transitions, G2178A and C2254T, that do not alter amino acid residues.

Mutations in 3 nuclear genes, ANT1, C10orf2, and POLG, have been associated with adPEO.^{4- 7,12- 14} Adenine nucleotide translocator-1, the gene responsible for the chromosome 4q35–linked form of adPEO,¹⁵ encodes an isoform of the adenosine triphosphate–adenosine diphosphate translocator common to muscle, heart, and brain. Adenine nucleotide translocator-1 regulates the adenine nucleotide pool within mitochondria and is a structural element of the mitochondrial permeability transition pore, thus playing an important role in apoptosis mediated by mitochondria.⁵ Twinkle, the product of the C10orf2 gene, appears to be an adenine nucleotide–dependent mtDNA helicase.⁴ Twinkle alterations may enhance dNTP breakdown and impair mtDNA replication through nucleotide pool imbalances.⁴

The α subunit of polymerase γ, the product of the POLG gene, is responsible for the chromosome 15q22-26–linked form of adPEO.⁶ Polymerase γ is a heterodimer composed of a 140-kDa α subunit and an accessory protein of about 41 kDa (β subunit).¹⁶ It is part of a multienzymatic complex located within the inner mitochondrial membrane and required for mtDNA replication.¹⁷ The α subunit is catalytic and contains both polymerase and exonuclease activities, whereas the β subunit seems to enhance DNA binding and promote DNA synthesis.¹⁸ Mutations in the β subunit have not been described in humans, but a null mutation of this subunit causes early death in Drosophila melanogaster.¹⁹

The human enzyme has weak homology with family A of DNA polymerases.^20- 22 Sequences in higher eukaryotes differ from those in lower eukaryotes, and similarities are confined mainly to the the amino-terminal exonuclease domain involved in proofreading and to the COOH-terminal polymerase domain.²² A unique characteristic of human POLG is the presence of 10 glutamine repeats near the amino-terminal.²⁰ The identification of novel POLG mutations in 4 families confirms that defects of POLG are common autosomal causes of PEO.^6- 7

Patient 1 had a heterozygous Gly1076Val substitution (Figure 2A). Glycine in residue 1076 is well conserved throughout evolution and is located near the COOH-terminal polymerase domain, between motifs B and C.²⁰ Glycine and valine have different structural characteristics. The simpler structure of glycine and the minimum steric impediment of its lateral chains allow greater flexibility of protein structures than other amino acid residues have; therefore, a glycine-to-valine amino acid change is likely to alter the stability of the secondary structure in this important region. The heterozygous Pro587Leu mutation identified in patients 2 and 3 (Figure 2B and C) may produce a similar effect on the protein secondary structure because proline is structurally different from leucine and has a secondary amino group that maintains a rigid conformation and reduces the structural flexibility of polypeptidic chains. Proline 587 is located immediately adjacent to motif III of the exonuclease domain.²⁰ In some species, proline is substituted by glutamic acid or threonine but never by leucine.

Patient 4 was compound heterozygous for Ala889Thr and Arg579Trp substitutions (Figure 2D and E). The alanine at residue 889 was also conserved and located exactly in the middle of motif A of the polymerase domain.²⁰ Although the alanine-to-threonine amino acid change is mild, its pathogenicity is probably enhanced by the association with the Arg579Trp change. Arginine 579 is relatively well conserved through evolution, although in some species it has been replaced by a lysine residue. Both arginine and lysine contain positively charged lateral groups, while tryptophan has an aromatic side group. Neither mutation produces clinical effects in heterozygosity, but when both mutations coexist in compound heterozygotes, they appear to have deleterious effects on POLG function.

Most mutations described in POLG affect functional exonuclease or polymerase domains.^6- 7 Two of the 4 novel mutations described here are located in exon 10, outside functional domains, which shows that the protein region encoded by exon 10 and adjacent to the exonuclease domain is important for the stability and correct conformation of the human protein.

Authors of in vitro studies have demonstrated that mutations in POLG can promote accumulation of mtDNA mutations, deletions, and depletion,^20,23 but the underlying mechanisms are unknown. Because Y955C mutated POLG has an enhanced base substitution error rate, Ponamarev et al²³ proposed that the mutated protein could promote deletions between direct repeats of mtDNA. A misinsertion event after correct synthesis of a repeat sequence of mtDNA could be followed by strand slippage between the direct repeats, thereby creating a matched DNA terminus at the downstream template sequence.²³

The results of this study can be used to confirm the genetic and clinical heterogeneity of diseases caused by POLG mutations. While mutations in ANT1 and C10orf2 have been associated with dominant or sporadic cases of PEO,^{3- 5,12- 14} mutations in POLG can cause either adPEO or arPEO.^6- 8 The clinical phenotypes of patients with POLG mutations appeared to be both more heterogeneous and more severe than those of patients with ANT1 and C10orf2 mutations.^6- 8,24 In addition to PEO, symptoms included psychiatric disorders, dysphagia, dysphonia, facial diplegia, neuropathy, ataxia, extrapyramidal syndromes, and profound muscle weakness.^7,24

In contrast, psychiatric disorders, neuropathy, and a motor neuron disease–like scenario were described in some cases of C10orf2 mutations,^4,14,25 whereas mutations in ANT1 caused a relatively homogeneous phenotype, usually limited to adPEO, ptosis, and muscle weakness^3,5,12- 13; only 1 family with a bipolar affective disorder and ANT1 mutation has been recently described.²⁶ In our patients with POLG mutations, the gastrointestinal symptoms resembled those described in mitochondrial neurogastrointestinal encephalomyopathy, an autosomal recessive disorder with multiple deletions and depletion of mtDNA caused by mutations in the gene encoding thymidine phosphorylase.²⁷ Hypogonadism, another clinical feature recently associated with POLG dysfunction,²⁸ was present in patient 3. There was both interfamilial and intrafamilial heterogeneity in severity and age at onset of symptoms. For example, in families 2 and 3, the parents were reportedly healthy (Figure 1B and C), but a maternal aunt was affected in family 2 (Figure 1B), which suggests that the Pro587Leu mutation has variable penetrance. Our data agree with those of a previous report that also showed variable penetrance, evidence of anticipation, and variable clinical expression of POLG mutations.⁷

We show that POLG mutations account for a substantial proportion of patients with PEO and multiple mtDNA deletions (13% of all cases; 17% of familial cases). However, the proportion of patients with familial autosomal PEO with POLG mutations in our series is lower than that in a previous study from Europe (46% [13 of 28]).⁷ This smaller number may be because of differences in the ethnic backgrounds of the 2 series of patients.

We suggest screening of POLG in patients with familial PEO with multiple mtDNA deletions, especially in patients with multisystemic involvement. However, it is also important to note that the genetic abnormalities underlying many cases of adPEO and arPEO remain unknown, which indicates that other genes are involved in the cause of these syndromes and remain to be identified.

Corresponding author and reprints: Salvatore DiMauro, MD, Department of Neurology, 4-420 Columbia University College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032 (e-mail: sd12@columbia.edu).

Accepted for publication February 13, 2003.

Author contributions: Study concept and design (Drs Filosto, Mancuso, Shanske, Hirano, and DiMauro); acquisition of data (Drs Filosto, Mancuso, Nishigaki, Harati, Gooch, Mankodi, Bayne, and Bonilla and Ms Pancrudo); analysis and interpretation of data (Drs Filosto, Mancuso, Shanske, Hirano, and DiMauro); drafting of the manuscript (Drs Filosto and DiMauro and Ms Pancrudo ); critical revision of the manuscript for important intellectual content (Drs Mancuso, Nishigaki, Harati, Gooch, Mankodi, Bayne, Bonilla, Shanske, Hirano, and DiMauro); study supervision (Drs Filosto, Hirano, and DiMauro).

This study was supported by National Institutes of Health grants NS11766 and PO1HD 32062 and by a grant from the Muscular Dystrophy Association; Department of Neurological Sciences and Vision, University of Verona, Italy (Dr Filosto); and Department of Neurosciences, University of Pisa, Italy (Dr Mancuso).